The present invention concerns processes for the recombinant production of S-layer proteins and modified S-layer proteins in gram-negative host cells.
Crystalline bacterial cell surface layers (S-layers) form the outermost cell wall component in many eubacteria and most of the archaebacteria (Sleytr et al. (1988), Crystalline Bacterial Cell Surface Layers, xe2x80x9cSpringer Verlag Berlinxe2x80x9d; Messner and Sleytr, Adv. Microb. Physiol. 33 (1992), 213-275). Most of the presently known S-layer proteins are composed of identical proteins or glycoproteins which have apparent molecular weights in the range of 40,000 to 220,000. The components of S-layers are self-assembling and most of the lattices have an oblique (p2), quadratic (p4) or hexagonal (p6) symmetry. The functions of bacterial S-layers are still not completely understood but due to their location on the cell surface the porous crystalline S-layers probably serve mainly as protective coatings, molecular sieves or to promote cell adhesion and surface recognition.
Genetic data and sequence information are known for various S-layer genes from microorganisms. A review may be found in Peyret et al., Mol. Microbiol. 9 (1993), 97-109. Explicit reference is made to these data. The sequence of the sbsA gene coding for the S-layer protein of B.stearothermophilus PV72 and a process for cloning it are stated in Kuen et al. (Gene 145 (1994), 115-120). B.stearothermophilus PV72 is a gram-positive bacterium which is covered with a hexagonally arranged S-layer. The main component of the S-layer is a 128 kd protein which is the most frequent protein in the cell with a proportion of about 15% relative to the total protein components. Various strains of B.stearothermophilus have been characterized which differ with regard to the type of the S-layer lattice, the molecular weight and glycosilation of the S-layer components (Messner and Sleytr (1992), supra).
The German Patent Application P 44 25 527.6 discloses the signal peptide-coding section of the S-layer gene from B.stearothermophilus and the amino acid sequence derived therefrom. The cleavage site between the signal peptide and the mature protein is located between position 30 and 31 of the amino acid sequence. The signal peptide-coding nucleic acid can be operatively linked to a protein-coding nucleic acid and can be used for the recombinant production of proteins in a process in which a transformed host cell is provided, the host cell is cultured under conditions which lead to an expression of the nucleic acid and to production and secretion of the polypeptide coded thereby and the resulting polypeptide is isolated from the culture medium. Prokaryotic organisms are preferably used as host cells in particular gram-positive organisms of the genus bacillus.
Surprisingly it was found that the recombinant production of S-layer proteins is not only possible in gram-positive prokaryotic host cells but also in gram-negative prokaryotic host cells. In this case the S-layer protein is not formed in the interior of the host cell in the form of ordered inclusion bodies but rather unexpectedly in the form of ordered monomolecular layers.
Hence one subject matter of the present invention is a process for the recombinant production of S-layer proteins characterized in that (a) a gram-negative prokaryotic host cell is provided which is transformed with a nucleic acid coding for an S-layer protein selected from (i) a nucleic acid which comprises the nucleotide sequence shown in SEQ ID NO. 1 from position 1 to 3684 optionally without the section coding for the signal peptide, (ii) a nucleic acid which comprises a nucleotide sequence corresponding to the nucleic acid from (i) within the scope of the degeneracy of the genetic code and (iii) a nucleic acid which comprises a nucleotide sequence which hybridizes with the nucleic acids from (i) or/and (ii) under stringent conditions; (b) the host cell is cultured under conditions which lead to an expression of the nucleic acid and to production of the polypeptide coded thereby and (c) the resulting polypeptide is isolated from the host cell.
The term xe2x80x9cstringent hybridizationxe2x80x9d is understood within the sense of the present invention to mean that a hybridization still also occurs after washing at 55xc2x0 C., preferably 60xc2x0 C. in an aqueous low salt buffer (e.g. 0.2xc3x97 SSC) (see also Sambrook et al. (1989), Molecular Cloning. A Laboratory Manual).
The process according to the invention is carried out in gram-negative prokaryotic host cells. In this process an ordered S-layer protein structure is surprisingly obtained in the cell interior. Enterobacteria, in particular E. coli, are preferably used as host cells.
The E. coli strain pop2135 which was deposited on 31.01.1996 at the xe2x80x9cDeutsche Sammlung von Mikroorganismen und Zelikulturen GmbHxe2x80x9d, Mascheroder Weg 1b, D 38124 Braunschweig under the file number DSM 10509 is particularly preferred
The process according to the invention can also be used to isolate recombinant S-layer proteins. For this one uses a nucleic acid coding for the S-layer protein which contains one or several insertions which code for peptide or polypeptide sequences. These insertions can, on the one hand, only code for peptides with a few amino acids e.g. 1-25 amino acids. On the other hand, the insertions can also code for larger polypeptides of for example up to 1000 amino acids and preferably up to 500 amino acids without loss of the ability of the S-layer protein to form a correctly folded structure. In addition to the insertions the recombinant S-layer protein can also have amino acid substitutions, in particular substitutions of individual amino acids in the region of the insertion sites as well as optionally deletions of individual amino acids or short amino acid sections of up to 30 amino acids.
Regions between the positions 1-1200 and 2200-3000 of the nucleotide sequence shown in SEQ ID NO.1 are preferred as insertion sites for polypeptide-coding sequences. Particularly preferred insertion sites are the NruI cleavage site at position 582, the PvuII cleavage site at position 878, the SnaB-I cleavage site at position 917, the PvuII cleavage site at position 2504 and the PvuII cleavage site at position 2649. It was already possible to demonstrate the insertion of a nucleic acid coding for streptavidin into the NruI cleavage site at position 581.
The peptide or polypeptide-coding insertions are preferably selected from nucleotide sequences which code for cysteine residues, regions with several charged amino acids, e.g. Arg, Lys, Asp or Glu, or Tyr residues, DNA-binding epitopes, antigenic, allergenic or immunogenic epitopes, metal-binding epitopes, streptavidin, enzymes, cytokines or antibody-binding proteins.
A particularly preferred example of an insertion into the nucleic acid coding for the S-layer protein is a nucleotide sequence coding for streptavidin. In this manner it is possible to obtain universal carrier molecules which are suitable for coupling biotinylated reagents and for detection in immunological or hybridization test procedures.
A further preferred example of insertions are antigenic, allergenic or immunogenic epitopes e.g. epitopes from pathogenic microorganisms such as bacteria, fungi, parasites etc. and viruses, or epitopes from plants or epitopes against endogenous substances e.g. cytokines as well as against toxins in particular endotoxins. Particularly preferred examples of immunogenic epitopes are epitopes from herpes viruses such as the herpes virus 6 or pseudorabies virus (Lomniczi et al., J. Virol. 49 (1984), 970-979), in particular epitopes from the genes gB, gC or/and gD, or foot-and-mouth disease virus (FMDV), in particular epitopes from the gene sections which code for VP1, VP2 or/and VP3. The immunogenic epitopes can be selected such that they promote an antibody-mediated immune reaction or/and the production of a cellular immune reaction e.g. by stimulation of T cells. Examples of suitable allergenic epitopes are birch pollen allergens e.g. Bet v I (Ebner et al., J. Immunol. 150 (1993) 1047-1054). Antigenic epitopes are additionally particularly preferred which are able to bind and filter out endogenous or exogenous substances such as cytokines or toxins from serum or other body fluids. Such epitopes can include components of cytokine or toxin receptors or of antibodies against cytokines or toxins.
On the other hand the insertions can also code for enzymes. Preferred examples are enzymes for the synthesis of polyhydroxybutyric acid e.g. PHB synthase.
Incorporation of PHB synthase into the S-layer can lead to the formation of a molecular spinning nozzle under suitable conditions when the substrate hydroxybutyric acid is provided. A further preferred example of an enzyme is bacterial luciferase. In this case when the enzyme substrate, an aldehyde, is supplied and 02 is present, a molecular laser can be obtained.
Insertions are likewise preferred which code for cytokines such as interleukins, interferones or tumour necrosis factors. These molecules can for example be used in combination with immunogenic epitopes to prepare vaccines.
Finally insertions are also preferred which code for antibody binding proteins such as protein A or protein G or for DNA-binding or/and metal-binding epitopes such as the leucine zipper, zinc finger etc.
Thus for the first time a cell is provided by the present invention which contains immobilized recombinant polypeptides in a native form e.g. active enzymes in the cytoplasm. In this manner 50,000-200,000 e.g. ca. 100,000 recombinant molecules can be immobilized per m2 recombinant S-layer. Up to 3000 m2 S-layer can be obtained per kg recombinant E. coli cells.
In the method according to the invention the nucleic acid coding for the S-layer protein is preferably used in operative linkage with a nucleic acid coding for a signal peptide of gram-positive bacteria i.e. the signal peptide-coding nucleic acid is located on the 5xe2x80x2 side of the S-layer protein-coding nucleic acid. Surprisingly it was found that the presence of such signal peptide sequences, which are not cleaved in the gram-negative host cells used in the invention, can improve the stability of the S-layer structures. The nucleic acid coding for the signal peptide particularly preferably comprises (a) the signal peptide-coding section of the nucleotide sequence shown in SEQ ID NO. 1, (b) a nucleotide sequence corresponding to the sequence from (a) within the scope of the degeneracy of the genetic code or/and (c) a nucleotide sequence which is at least 80% and in particular at least 90% homologous to the sequences from (a) or/and (b).
Yet a further subject matter of the present invention is a nucleic acid which codes for a recombinant S-layer protein and is selected from (i) a nucleic acid which comprises the nucleotide sequence shown in SEQ ID NO.1 from position 1 to 3684 optionally without the signal peptide-coding section (ii) a nucleic acid which comprises a nucleotide sequence corresponding to a nucleic acid from (i) within the scope of the degeneracy of the genetic code and (iii) a nucleic acid which comprises a nucleotide sequence which hybridizes under stringent conditions with the nucleic acids from (i) or/and (ii).
The coding nucleotide sequence of the S-layer gene sbsA from B.stearothermophilus including the signal peptide-coding section is shown in SEQ ID NO. 1. The signal peptide-coding section extends from position 1 to 90 of the nucleotide sequence shown in SEQ ID NO. 1. The section coding for the mature SbsA polypeptide extends from position 91 to 3684.
The sbsA gene of B.stearothermophilus codes for a protein with a total of 1228 amino acids including an N-terminal signal peptide with 30 amino acids (SEQ ID NO. 2). The cleavage site between the signal peptide and the mature protein is located between position 30 and 31 of the amino acid sequence. The signal peptide has a basic amino-terminal domain followed by a hydrophobic domain.
Sequence comparisons with other signal peptides indicate a certain homology to signal peptides of extracellular proteins in bacilli such as alkaline phosphatase and neutral phosphatase of B.amyloliquefaciens (Vasantha et al., J. Bacteriol. 159 (1984), 811-819) as well as with the signal peptides for the B.sphaericus gene 125 (Bowditch et al., J. Bacteriol. 171 (1989), 4178-4188) and the OWP qene of B.brevis (Tsuboi et al., J. Bacteriol. 168 (1986), 365-373).
A further subject matter of the present invention is a recombinant vector which contains at least one copy of a nucleic acid according to the invention. The vector is preferably replicatable in prokaryotes. The vector is particularly preferably a prokaryotic plasmid.
Yet a further subject matter of the present invention is a host cell which is transformed with a nucleic acid or a recombinant vector according to the present invention. The cell is preferably a gram-negative prokaryotic organism and most preferably an E. coli cell. The cell according to the invention can contain a recombinant S-layer structure in its interior. Methods for the transformation of cells with nucleic acids are general state of the art (cf. Sambrook et al., supra) and therefore do not need to be elucidated.
Yet a further subject matter of the present invention is a recombinant S-layer protein which contains at least one peptide insertion or/and polypeptide insertion within the amino acid sequence shown in SEQ ID NO. 2. Preferred examples of peptide insertions and polypeptide insertions have already been elucidated.
A recombinant S-layer structure can be assembled from recombinant S-layer protein molecules according to the invention which contain at least one recombinant S-layer protein according to the invention as a subunit. Furthermore it is preferred that the S-layer structure according to the invention also contains non-modified S-layer proteins as diluent molecules. The non-modified S-layer proteins are preferably present in a molar proportion of 10-99% relative to the total S-layer proteins.
The S-layer structure according to the invention can comprise several layers that are covalently linked together or by means of affinity binding. Covalent linkages can for example be introduced by insertions of cysteine residues and a subsequent formation of cystine bridges. Linkages by affinity binding comprise for example antibody-antigen, antibody-protein A or antibody-protein G or streptavidin-biotin interactions.
S-layer structures which contain recombinant S-layer proteins can optionally also be prepared in a carrier-bound form. For this the S-layer structure can be reassembled from individual units in the presence of a peptidoglycan carrier to for example produce peptido-glycan layers which are coverged on one or on both sides with an S-layer structure. Another method of preparing carrier-bound S-layer structures is to produce an S-layer layer at an interface between two media e.g. water/air and to immobilize this layer on a solid phase e.g. a filter membrane (cf. e.g. Pum and Sleytr (1994), Thin Solid Films 244, 882-886; Kupcu et al., (1995), Biochim. Biophys. Acta 1235, 263-269).
The recombinant S-layer proteins and S-layer structures according to the invention are suitable for a multitude of applications. An application as a vaccine or adjuvant is particularly preferred in which case recombinant S-layer proteins are used which contain immunogenic epitopes of pathogens and/or endogenous immuno-stimulatory polypeptides such as cytokines. In this application it is not absolutely necessary to purify the recombinant S-layer proteins. Instead they can for example be used in combination with a bacterial ghost which optionally contains additional immunogenic epitopes in its membrane.
The preparation of suitable xe2x80x9cbacterial ghostsxe2x80x9d is described for example in the International Patent application PCT/EP91/00967 to which reference is herewith made. In this application modified bacteria are disclosed which are obtainable by transformation of a gram-negative bacterium with the gene of a lytically active membrane protein from bacteriophages, with the gene of a lytically active toxin release protein or with genes which contain partial sequences thereof which code for lytic proteins, culturing the bacterium, expression of this lysis gene and isolation of the resulting bacterial ghost from the culture medium.
A recombinant protein, which is obtainable by expression of a recombinant DNA in these gram-negative bacteria, can be bound to the membrane of these bacteria as described in the European Patent 0 516 655. This recombinant DNA comprises a first DNA sequence which codes for a hydrophobic, non-lytically active membrane-integrating protein domain which has an a-helical structure and is composed of 14-20 amino acids which can be flanked N- and C-terminally by 2-30 arbitrary amino acids in each case. A second DNA sequence is in operative linkage with this first DNA sequence which codes for a desired recombinant protein. Furthermore the gram-negative bacterium contains a third DNA sequence which is under a different control from the first and second DNA sequences and codes for a lytically active membrane protein from bacteriophages or a lytically active toxin release protein or for their lytically active components. So-called xe2x80x9cbacterial ghostsxe2x80x9d are obtained by expression and lysis of such recombinant gram-negative bacteria which contain an intact surface structure with immunogenic epitopes bound to the surface.
When these bacterial ghosts are combined with recombinant S-layers according to the invention vaccines and adjuvants can be produced which have particularly advantageous properties.
A further particularly preferred application for recombinant S-layer proteins and S-layer structures is their use as an enzyme reactor. Such an enzyme reactor can for example be formed by a cell which contains a recombinant S-layer structure according to the invention in its interior. On the other hand the enzyme reactor can also be formed from isolated and in vitro reassembled S-layer structures or combinations of various S-layer structures.
It was found that the gram-positive bacterium B.stearothermophilus PV72 has an additional S-layer protein in addition to SbsA which is subsequently denoted as SbsB (Sara and Sleytr (1994), J. Bacteriol. 176, 7182-7189). It was possible to isolate and characterize the sbsB gene by amplification using suitable nucleic acid primers. The coding nucleotide sequence of the S-layer gene sbsB from B.stearothermophilus including the signal peptide-coding section which extends from position 1 to 93 of the nucleic acid sequence is shown in SEQ ID NO.5. The amino acid sequence derived therefrom is shown in SEQ ID NO.6. The sbsB gene codes for a protein with a total of 921 amino acids including an N-terminal signal peptide with 31 amino acids.
One subject matter of the present invention is hence a nucleic acid which codes for an S-layer protein and is selected from
(i) a nucleic acid which comprises the nucleotide sequence from position 1 to 2763 shown in SEQ ID NO.5 optionally without the signal peptide-coding section,
(ii) a nucleic acid which comprises a nucleotide sequence corresponding to the nucleic acid from (i) within the scope of the degeneracy of the genetic code and
(iii) a nucleic acid which comprises a nucleotide sequence that hybridizes with the nucleic acids from (i) or/and (ii) under stringent conditions.
As in the case of the sbsA gene, it is also possible to insert at least one nucleic acid insertion coding for a peptide or polypeptide into the sbsB gene within the region coding for the S-layer protein. With regard to preferred examples of insertions in the sbsB gene reference is made to the previous statements regarding the sbsA gene.
Yet a further subject matter of the present invention is a vector which contains at least one copy of an sbsB gene optionally containing an insertion. This vector can be replicated in eukaryotes, prokaryotes or in eukaryotes and prokaryotes. It can be a vector that can be integrated into the genome of the host cell or a vector which is present extrachromosomally. The vector according to the invention is preferably a plasmid in particular a prokaryotic plasmid.
Yet a further subject matter of the present invention is a host cell which is transformed with an sbsB gene wherein the sbsB gene optionally can contain an insertion. The host cell can be a eukaryotic as well as a prokaryotic cell. The cell is preferably a prokaryotic organism. Gram-positive organisms e.g. organisms of the genus bacillus as well as gram-negative organisms such as enterobacteria in particular E. coli are preferred. Methods for transforming eukaryotic and prokaryotic cells with nucleic acids are known and therefore do not need to be elucidated in detail.
The present invention also concerns an SbsB protein i.e. an S-layer protein which is coded by a nucleic acid as defined above. Recombinant SbsB proteins are particularly preferred which contain one or several peptide or/and polypeptide insertions within the sbsB sequence. The SbsB part of a polypeptide according to the invention particularly preferably has a homology of at least 80% and in particular of at least 90% to the amino acid sequence shown in SEQ ID NO.6.
A recombinant S-layer structure can also be assembled from the recombinant SbsB-S-layer protein molecules analogous to the recombinant SbsA-S-layer structure. In this structure the non-modified S-layer proteins are preferably present in a molar proportion of 10-99% relative to the total S-layer proteins.
The applications for the recombinant SbsB-S-layer proteins and S-layer structures according to the invention also correspond to the applications for SbsA mentioned above. In this connection its use as a vaccine or adjuvant or as an enzyme reactor is noteworthy.
Recombinant S-layer proteins are obtainable by a process in which
(a) a host cell is provided which contains a nucleic acid coding for an S-layer protein which contains a peptide-coding or polypeptide-coding insertion within the region coding for the S-layer protein,
(b) the host cell is cultured under conditions which lead to an expression of the nucleic acid and to production of the polypeptide coded by it and
(c) the resulting polypeptide is isolated from the host cell or from the culture medium.
In a first preferred embodiment of this process a recombinant SbsA-S-layer protein is prepared i.e. the nucleic acid coding for the recombinant S-layer protein is selected from
(i) a nucleic acid which comprises the nucleotide sequence from position 1 to 3684 shown in SEQ ID NO.1 optionally without the signal peptide-coding section,
(ii) a nucleic acid which comprises a nucleotide sequence corresponding to the nucleic acid from (i) within the scope of the degeneracy of the genetic code and
(iii) a nucleic acid which comprises a nucleotide sequence which hybridizes with the nucleic acids from (i) or/and (ii) under stringent conditions.
In a second preferred embodiment a recombinant SbsB-S-layer protein is prepared i.e. the nucleic acid coding for the recombinant S-layer protein is selected from
(i) a nucleic acid which comprises the nucleotide sequence from position 1 to 2763 shown in SEQ ID NO.5 optionally without the signal peptide-coding section,
(ii) a nucleic acid which comprises a nucleotide sequence corresponding to the nucleic acid from (i) within the scope of the degeneracy of the genetic code and
(iii) a nucleic acid which comprises a nucleotide sequence which hybridizes with the nucleic acids from (i) or/and (ii) under stringent conditions.
In addition to the recombinant SbsA and SbsB-S-layer proteins from B.stearothermophilus it is, however, also possible to prepare recombinant S-layer proteins from other organisms (cf. e.g. Peyret et al., (1993), supra).
The recombinant S-layer proteins can on the one hand be produced in a heterologous host cell i.e. in a host cell which originally contains no S-layer gene. Examples of such heterologous host cells are gram-negative prokaryotic organisms such as E. coli. 
However, the heterologous expression of S-layer proteins can also take place in gram-positive prokaryotic organisms such as B. subtilis. For this integration vectors are preferably used which contain a native or/and a recombinant S-layer gene. When the native signal sequences are used the S-layer proteins are secreted into the culture supernatant.
However, it is often preferable to produce the recombinant S-layer proteins in homologous host cells i.e. host cells which originally contain a natural S-layer gene. In one embodiment of this homologous expression the recombinant S-layer gene is introduced into the host cell in such a way that the host cell is still able to express a further S-layer gene which codes for a non-modified S-layer protein. The non-modified S-layer protein is preferably capable of forming an S-layer structure that is compatible with the recombinant S-layer protein. An example of this embodiment of homologous expression is a B.stearothermophilus PV72 cell which contains intact natural sbsA genes or/and sbsB genes and is transformed with a plasmid which contains a recombinant S-layer gene.
In a second embodiment the homologous expression can occur in a host cell in which the intact S-layer gene originally present has been inactivated. Consequently in this embodiment no further S-layer gene is expressed in the host cell which codes for a non-modified S-layer protein which is able to form a compatible S-layer structure with the recombinant S-layer protein. A specific example of such a host cell is a B.stearothermophilus PV72 cell in the genome of which a gene coding for a recombinant S-layer protein has been introduced, e.g. by homologous recombination, which replaces the original S-layer gene. A further example of such a host cell is a B.stearothermophilus cell in which the native S-layer gene has been inactivated e.g. by site-specific mutagenesis or/and homologous recombination and is transformed with a vector containing a recombinant S-layer gene.
Gram-positive prokaryotic organisms are usually used as host cells for the homologous expression of recombinant S-layer genes. B.stearothermophilus PV72 is particularly preferred as a host cell which can be cultured at a high temperature in a defined synthetic medium (Schuster et al., (1995), Biotechnol. and Bioeng. 48: 66-77).